The purpose of this proposal is to study the biochemical events mediating intracellular thyroid hormone metabolism, in particular thyroxine (T4) to triiodothyronine (T3) conversion and T3 degradation in both the kidney and brain. Previous studies have indicated that conversion of T4 and T3 is a critical, if not obligatory, step in the activation of T4 in both man and animals. These studies have also revealed changes in T4 and T3 conversion in all tissues in response to various physiological perturbations but the mechanism for these changes is unknown. The brain and kidney serve as models of two general types of intracellular iodothyronine metabolism; one, brain (like pituitary), efficiently utilizes locally generated T3. The other, kidney (together with the liver), provides most of the T3 found in the circulation. Iodothyronine 5'-deiodinase, the enzyme catalizying T4 to T3 conversion will be isolated from detergent extracts of kidney membranes and phospholipid requirements, hydrodynamic and immunological properties of both the crude extracts and purified enzyme, obtained by affinity chromatography, investigated. The role of enzyme sulfhydryls will be characterized by chemical modification and methods for selective labeling of enzyme polypeptides evaluated. The analogous enzyme in rat brain will be compared and contrasted to that in the kidney with respect to subcellular distribution, kinetics of the enzyme reaction, hydrodynamic and immunological properties. The subcellular distribution in brain of tyrosyl ring deiodinase, the enzyme which catalyzes T3 degradation, will be determined and kinetic properties evaluated. The effects of thyroid hormone excess and deficiency on both T4 to T3 conversion and T3 degradation in the brain will be used to clarify the tissue factors which may regulate iodothyronine metabolism and could explain the often divergent response to T4 to T3 converting activity to the same physiological stimulus in these two tissues. Comparisons between the functional and physical properties of these enzymes will provide a basis for understanding the mechanisms by which mammalian tissues modulate thyroid hormone.